Abstract This MIRA application combines three NIGMS awards R01GM070837, 1R01GM110639, and 5R01GM108487, studying the tethering and integration the murine leukemia virus (MLV) preintegrative complex (PIC). Gammaretroviruses require cells to undergo mitosis for integration and preferentially integrate within active promoters regions, proximal to transcriptional start sites (TSS) and CpG islands. The requirement for mitosis limits the use of these vectors to dividing cells. The site of integration is linked to the oncogenic potential of the virus and thus its pathogenesis and use as a gene delivery vector. Studies are focused on three targeted areas. The p12 protein is required for tethering the PIC to mitotic chromosomes. Recent analysis of MLV p12 tethering properties has revealed a novel mechanism of suppression of tethering localizing in the N-terminus of the p12 protein. A model is proposed that incorporates an orientation reversal from the precursor Gag protein to that found in the PIC that transitions through an intermediate in which the tethering domain is inhibited by N- and C-terminal charge interactions. Experiment testing this model will involve biochemical and structural approaches, including NMR analysis of the p12 tethering domain. Our collaborative research has identified the host BET family of proteins, specifically the Brd 2,3, and 4 proteins as interactors with the MLV IN. The interaction between the MLV IN C- terminal tail peptide and the ET domain influences the integration into TSS and CpG islands. Studies proposed build on this newly defined viral-host interaction. Building on the solution structure of the MLV IN CTD from our laboratory, experiments are aimed at defining the structural data of larger complexes including the MLV IN CTD:Brd3 ET complex, and the domains of IN bound to DNA. The ability of the MLV IN protein to compete with known ET domain interactors will be studied. Next-generation sequencing and bioinformatics will be used to probe a panel of IN proteins for their target-site preferences, both locally at the site of integration as well as globally within the human genome. The recognition of the target DNA within the nucleosome structure is examined. These studies will be combined with two emerging areas in our laboratory. The first develops MLV based virus-like particles (VLP) for the delivery of proteins, rather than genes into cells. The second area of research develops a modified Env system that allows entry through scFvs and monobodies as targeting moieties. This system could be generalized and expanded to address a wide range of applications and diseases.